Chemical modification of cholecystokinin‐A receptors in rat pancreatic membranes

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Abstract
Chemical modification of amino acids was used to probe the molecular structure of the cholecystokinin‐A (CCK‐A) receptor on rat pancreatic membranes. Radioligand binding studies with [3H]N‐(2,3‐dihydro‐l‐methyl‐2‐oxo‐5‐phenyl‐lH‐l,4‐benzodiaz epin‐3‐yl)1H‐indole‐2‐carboxamide[(±)‐[3H]L‐364,718], a tritiated highly potent CCK‐A receptor antagonist, enabled the evaluation of the effects caused by the modifying reagents. The apparent fragility of the receptor protein necessitated the development of a modification procedure without wash and centrifugation steps. Treatment of a concentrated membrane preparation with the group‐specific agents N‐ethylmaleimide, phenylglyoxal and diethylpyrocarbonate, subsequent dilution and incubation at lower temperatures (20°C instead of the more generally used 37°C) proved successful. All modifiers affected the binding characteristics for both agonists and antagonists considerably. CCK‐A receptor coupling to guanosine‐nucleotide‐binding proteins was substantially diminished upon modification with N‐ethylmaleimide and diethylpyrocarbonate, as could be concluded from the effects on the (±)‐[3H]L‐364,718 displacement by the cholecystokinin C‐terminal octapeptide (CCK‐8). The ligand‐binding site was affected by all three reagents, as could be inferred from the specific protection obtained with the CCK‐A receptor antagonist, lorglumide. It therefore appears that sulfhydryl, arginyl, and histidyl residues form an essential part of the ligand‐binding domain on the CCK‐A receptor and that sulfhydryl and histidyl residues are also involved in the signal‐transduction pathway.